CYP Inhibition, LC-MS/MS Detection (IC50 Determination)

Assay System: 

  • Human liver microsomes, pooled from 50 donors.
  • Inhibition potential of test compound measured upon incubation with human liver microsomes at different compound concentrations. Reversible inhibition is determined by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.

Assay Conditions:

Test Compound Concentration Eight concentrations ranging from 0.05 µM to 100 µM
Percent Organic Solvent in Incubation 2% acetonitrile
CYP Isoforms Available for Testing CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
Incubation Time 10-20 minutes (depending on CYP isoform)
pH 7.4
Temperature 37 °C
Analysis LC-MS/MS
Number of Repeats 2

Deliverable: IC50 of test compound and positive control compound.

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.

CYP Isoform Probe Substrate Monitored Metabolite Positive Control Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
2B6 Bupropion OH Bupropion Ticlopidine HCl
2C8 Amodiaquine Desethylamodiaquine Montelukast
2C9 Tolbutamide 4’-OH Tolbutamide Sulfaphenazole
2C19 S-Mephenytoin 4’-OH Mephenytoin (+)-N-3-Benzylnirvanol
2D6 Dextromethorphan Dextrorphan-D-Tartrate Quinidine
3A4 Testosterone 6β-OH Testosterone Ketoconazole
3A4 Midazolam 1’-OH Midazolam Ketoconazole
*If you choose "Other", please use the comments section below to request your desired system.